1. Technical Field
The present invention relates to a method for analyzing nucleic acid, specially to a blotting method for rapidly analyzing nucleic acid in which the nucleic acid to be analyzed is directly transferred to a substrate, and the time for nucleic acid hybridization is shortened.
2. Technical Background
In the conventional gene cloning, in order to determine whether the gene or DNA fragment is correctly cloned in the carrier, the gel electrophoresis may be performed to analyze the molecular weight of the DNA. In addition, another more precise means is by hybridizing a nucleic acid probe with the DNA to be determined to analyze whether the DNA to be determined comprises the desired gene or DNA fragment. Likewise, in order to study the gene expression, the length and expression amount of mRNA generated after transcription, is required to be detected after the expression of gene, by hybridizing a nucleic acid probe with the RNA and then analyzing the length or copy number of the RNA.
A blotting method generally comprises the steps of absorbing the nucleic acid to be analyzed on a membrane, hybridizing and pairing the nucleic acid with a probe having specificity, and then presenting the result of the hybridization by the probe-marked present molecule in a presenting method such as coloring method, chemiluminescence method, radiography method and so on. The nucleic acid to be analyzed may be absorbed on the membrane by transferring from electrophoresis gel or directly dropping the nucleic acid to be analyzed on the membrane to make the nucleic acid penetrated therein. A blotting method in which a DNA sample transferred to the membrane by electrophoresis gel is hybridized with the probe is referred to as Southern blotting method. A blotting method in which a RNA sample transferred is hybridized is referred to as Northern blotting method. A blotting method in which the nucleic acid to be analyzed is directly dropped is referred to as dot-/slot-/spot blotting method according to the dropping area. The dot-/slot-/spot blotting method is usually applied in qualitative analysis or scores of analysis, because the analyzing time can be shortened due to the needlessness of electrophoresis for separation and transfer, and the cost is low due to the needlessness of electrophoresis and transferring devises and related agents.
Referring to FIG. 1, it is a flow chart showing the known dot blotting method. The whole blotting method comprises pretreatment of the membrane (step S10), treatment of the nucleic acid to be analyzed (step S20), hybridization of the nucleic acid (step S30) and detection of the hybridization signal (step S40). The membrane is firstly treated, the nucleic acid to be analyzed is dropped thereon, the nucleic acid is hybridized with a probe, and finally the hybridization signal is detected to finish the whole procedure of dot blotting.
The membrane pretreatment in step S10 comprises providing a membrane able to absorb the nucleic acid to be analyzed (step S11), and then soaking the membrane (step S12) to make the membrane be wet to further penetrate and absorb the nucleic acid to be analyzed. Currently conventional membrane includes nylon membrane and nitrocellulose membrane which is soaked completely in deionized water and then in 6 to 10 folds standard sodium citrate (SSC) for use, wherein 20 folds SSC comprises 3 M sodium chloride and 0.3 M sodium citrate of pH 7.0.
Prior to the hybridization, either DNA sample or RNA sample has to be subjected to denaturation to keep itself in a state of single chain, in order to pair with the probe of single chain. Accordingly, during treatment of the nucleic acid to be analyzed in step S20, the nucleic acid is prepared in step S21, denatured in step S22, and then dropped on the membrane in step S23. After being absorbed on the membrane, the nucleic acid to be analyzed is fixed thereon to prevent it from falling off in the subsequent hybridization process. The known fixing method for nucleic acid is to dry for 30 minutes to 2 hours at 80° C. in step S24, or to radiate with ultraviolet at 254 nm to form covalent crosslinkage between the nucleic acid molecules and cellulose of the membrane to fix the nucleic acid molecules thereon in step S25.
After being fixed on the membrane, the nucleic acid to be analyzed is then hybridized in step S30. The nucleic acid to be analyzed has not been fixed on some areas of the membrane where the nucleic acid can be still absorbed, and if these areas are not blocked, the added probe which is also nucleic acid will be non-specifically bonded to these areas of the membrane. Accordingly, prior to the hybridization, the step of prehybridization S31 has to be performed by adding a solution containing 5 folds SSC solution, 50% (v/v) formamide, 0.1% (w/v) sodium dodecyl sulfate (SDS) solution and 5% casein on the membrane and reacting them for 1 to 2 hours at 42° C. to block the areas where the nucleic acid to be analyzed has not been fixed.
Subsequently, in the hybridization step S32, the nucleic acid probe marked with enzyme or radioactive elements is added on the membrane having been subjected to the prehybridization, and is then reacted for more than 16 hours at 42° C. During this course, the probe can find and pair with the complementary nucleic acid to be analyzed. Then, in the washing step S33, the nucleic acid probe that is not paired will be washed off. During washing, a washing solution containing 2 folds SSC solution and 0.1% (w/v) SDS is used to wash twice at the ambient temperature and for 5 minutes each time. And then a washing solution containing 0.1 fold SSC solution and 0.1% (w/v) SDS is used to wash twice at 68° C. and for 15 minutes each time.
After the washing step S33, only the probes pairing with the nucleic acid to be analyzed remain on the membrane, and at this time the detection step S40 can be performed to detect the hybridization signal. A suitable detection method may be used to detect the hybridization signal according to the detecting molecules marked with the probe. The conventional detection method includes color reaction detection method S41 using the digoxigenin (DIG) system, radiography detection method S42 marking the radioactive element 32P or 35S, and chemiluminescence detection method S43 using HEX, Cy3 and Cy5.
It could be seen from the above steps that, in the known blotting method, it takes many procedures and long time to finish the membrane pretreatment (step S10), treatment of the nucleic acid to be analyzed (step S20), and hybridization of the nucleic acid (step S30), and it will take about 2 days to finish all the procedures including the detection of the hybridization signal (step S40). Such that the experiments of which the result is urgent to get can not be finished in a short time. In addition, if a lot of time and agents should be also used for some simple qualitative test of nucleic acid, the method will be not economical. Accordingly, it is required to develop a rapid blotting method in which the time and procedures for blotting analysis can be shortened and the background noise can be lowered. For either the simple test or scores of tests, the experiment time can be effectively shortened, and at the same time the cost of the consumed materials can be largely lowered.